GWI and CFS remain poorly understood illnesses with complex etiology. Though they show considerable overlap in clinical presentation there is mounting evidence supporting the involvement of characteristic immune and endocrine dysfunction in each illness. In this work we compared these sister illnesses and their cytokine expression profiles in both male and female subjects separately to explore the role of endocrine-mediated modulation of immune response. As persistent and disabling fatigue is a key presenting symptom in both illnesses, a graded exercise challenge was used to stimulate immune response and amplify cytokine expression in a clinically relevant way. Several cytokines are recognized as exercise-responsive or metabolically active in healthy individuals. These myokines include IL-1ra, 6, 8, 10, 15 and TNF-α [41, 42]. In this work IL-10 and IL-15 appear as illness markers in male subjects with expression levels suggesting a delayed or compromised anti-inflammatory response to exercise. The same would appear true of exercise response in female CFS subjects. Interestingly significant differences in the immune response to exercise between male and female populations were immediately observed even in the healthy control group. Healthy female subjects could be distinguished from their male counterparts by significantly elevated expression of IL-12 and IL-23. While male GWI subjects could be distinguished from control on the basis of cytokine expression at rest but not at peak effort, the opposite was true for female subjects. Moreover, female GWI and CFS subjects could be separated almost perfectly on the basis of IL-1b and IL-5 levels alone, albeit in a small pilot cohort (n = 10). Indeed, cytokine expression would typically trend lower than control in female CFS and higher than control in female GWI, reaching statistical significance for levels of IL-4, 5 and 8. In contrast, male GWI could not be distinguished easily from their CFS counterparts on the basis of minimally overlapping cytokine markers as these typically trended in the same direction compared to control. Separation of these subjects was achieved in previous work by our group  but this work made extensive use of cytokine response measured in supernatants isolated and treated in vitro with the mitogen phytohaemagglutinin (PHA), a stimulant of T cell activity. The current work would suggest these groups may share several features of cytokine co-expression in plasma and that the patterns and levels obtained in response to in vitro stimulation with a specific mitogen may be required to provide the resolution necessary to distinguish one set from the other unequivocally. Moreover, as in this previous analysis it may also be necessary to reconcile partially correlated cytokines and use these in conjunction with one another instead of truncating in order to converge on a set of minimally overlapping cytokines. This observation was further confirmed in our analysis of female CFS subjects. Separation of these subjects from healthy control improved dramatically when the requirement for mutual independence was relaxed and statistical corrections were made to include and reconcile the co-expressed markers IL-17 at peak effort and IL-10 at recovery. Likewise male CFS and GWI subjects could be separated almost completely when adjusting for the co-expression of TNFβ, IL-1β, 2 and 6 at rest and peak effort.
These differences not withstanding, in looking across all subject groups we found a recurrent theme consisting of cytokines directly or indirectly involved with Th17 activity . For example, IL-23 was a distinguishing feature of sex in healthy subjects. It was also a significant contributor to illness signature in both CFS and GWI, along with IL-17 across both sexes. In one subset or another, this was accompanied by contributions from IL-1b, IL-2 and IFNγ among others. In addition IL-6 and IL-8 contributed to differences between male and female subjects within each illness group. It is interesting to note that most of these cytokines were not differentially expressed across groups even though they contributed significantly to the classification of these subjects. IL-1, 6 and 23 are known inducers of Th17 response, which in turn is a producer of IFNγ . Conversely IL-2 is generally known as a Th17 antagonist . Indeed the expression of these cytokines is not as disparate as it may seem. For example, Liu et al. (2007)  describe a mechanism by which IL-1 induces the production of IL-23 via NF-kappa B activation, which in turn promotes the production of IL-6 and 8 in human fibroblast-like synoviocytes from rheumatoid arthritis patients. IL-10 was also selected across illnesses, particularly in male subjects, as an important contextual element for IL-23 expression. Work by Verreck et al. (2004)  has implicated the interplay between IL-23-producing type 1 macrophages and IL-10-producing type 2 macrophages in modulating immune response to mycobacteria.
Overlap between IL-23 as an illness marker and its contribution (along with family member IL-12) as a marker of gender-specific response to exercise suggests significant involvement of the sex hormone axis in modulating the IL-23/Th17/IL-17 immune response. For example there is evidence that progesterone may suppress Th1 and Th17 pathway activity and induce an anti-inflammatory response when present at pregnancy levels . Conversely progesterone has been shown to enhance Th2 and T regulatory (Treg) responses. In the current analysis the appearance of IL-4 and IL-5 as markers of GWI and CFS in the female subset further supports possible involvement of a sex hormones in modulating the balance between Th2 and Th17 response. Studies in human cell cultures and in mice have shown that human T cells exhibit a sex-specific difference in the production of IFNγ and IL-17 driven by androgen status and possibly the expression of peroxisome proliferator activated receptors PPARα and PPARγ . In fact, in animal models the disappearance of normal sex-driven differences in the gene regulatory network modulating Th17 immune response has been implicated recently in supporting an increased susceptibility to rheumatoid arthritis (RA) . Collectively these works and the analysis presented here suggest that imbalance in the sex hormone regulation of Th17 immune response may be a shared component of these illnesses.
In GWI specifically, recent work has pointed to disruption of normal cholinergic regulation of immune function as a possible result of exposure to battlefield agents [48, 49]. For example, mRNA levels of cytokines IL-6, IL-17 and IL-13 among others have been shown to respond readily to organophosphate exposure . Acetylcholine (ACh) is known to regulate T cell function by acting on the nicotinic (nAChR) and muscarinic (mAChRs) cholinergic receptors . In recent work, Qian et al. (2011)  demonstrated in culture that nicotinergic stimulation of CD4+/CD62L(L-selectin) + T cells upregulated IFNγ and down-regulated IL-17 secretion, whereas the muscarinic stimulation enhanced IL-10 and IL-17 while inhibiting INFγ secretion. Muscarinic acetylcholine signaling has also been found to promote IL-8 release via PKC, ERK1/2 and NF-ΚB pathways . Similarly nicotinic receptor (nAChR) antagonists, ERK1/2 inhibitors or intracellular calcium chelators reduced IL-8 secretion by human epithelial cells . Based on this finding, persistent cholinergic stimulation would support the increased levels of IL-8 observed here in both male and female GWI subjects. Increased nicotanergic activation would support an ACh-driven down-regulation of Th17 response evidenced by decreased IL-23 in female GWI subjects. Elevated IL-5 levels in this group would further support the suppression of IL-17 response as part of a sex hormone mediated shift towards a Th2 status. The absence of this added sex-specific driver of Th2 polarization might explain the more subdued trend towards Th17 suppression observed in male GWI subjects. It is important to note that the above-mentioned mechanisms were typically studied under specific experimental conditions whereas the immune signatures reported in this work were obtained in whole blood from human subjects. In order to obtain a more detailed understanding of the precise mechanisms that underlie these cytokine profiles it will be important to cast these in the context of the corresponding immune cell demographics using flow cytometric methods .
Though with some illness-specific variations, a continued trend away from Th2 polarity is observed in male CFS subjects with these subjects showing considerable overlap in cytokine expression with their GWI counterparts. We also found a decrease in Th2 polarity when comparing levels of IL-4 and IL-5 in female CFS with those in female GWI. In our earlier work with a much larger cohort of female CFS subjects (n = 40; mean age 53 ± 1.8 years) , we had found elevated levels of Th2 markers in female CFS subjects when compared to healthy controls. As discussed above, a potentially important contributor to this may be menopausal status since sex hormones can be important modulators of Th2 polarization [45, 56] and age has been shown to be an important contributor to increased IFNγ response in women . In comparison, the opposite trend in IFNγ response with age is typically true in men and occurs over a more narrow range. It is important to note therefore that this previous work with female CFS subjects was biased towards a post-menopausal population (n = 40; mean age 53 ± 1.8) whereas the smaller cohort used in this analysis would be more adequately described as peri-menopausal (n = 10; mean age 43 ± 2.8). Indeed when we compared healthy control groups across these studies we found significantly lower expression of IL-4 in the older control group (p = 0.003) while IL-4 levels in both the older and younger CFS groups were comparable (p = 0.49) (Additional file 3: Figure S1). Certainly the reduced size of the current cohort, in particular the number of female subjects, may have limited the statistical resolution for detecting differences in the expression levels of individual cytokines. However, the compatibility of healthy control groups should almost certainly be considered when comparing immune signatures across studies especially in women, as these control populations constitute a moving endocrine-immune benchmark. In addition to sex hormones, stress and growth hormones produced along the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-thyroidal (HPT) axes are also important immune modulators [58, 59]. Moreover, dysfunction of these axes has also been documented in these illnesses [6, 60]. Although in this study the immune-modulatory effects of these hormones were implicit to the cytokine signatures, their direct measurement would undoubtedly reduce unexplained variations in immune signature and potentially improve the resolution of a diagnostic panel for these illnesses.
These limitations notwithstanding, certain preliminary observations regarding the immune processes involved in these illnesses may still be made. In particular, it was interesting to note that the IL-23/Th17/IL-17 axis appears differentially modulated across sexes in healthy individuals and that different markers of this same axis appear to contribute to the delineation of GWI and CFS in a sex and illness-specific way. Our work across multiple cohorts has shown this theme of Th17 dysfunction to be highly conserved even though individual markers may not be. This work also suggests that more robust cytokine signatures may be obtained by accounting for sex-specific differences in endocrine-immune crosstalk and that future studies should consider male and female CFS and GWI subjects as 4 distinct groups for observational, mechanistic, diagnostic, and potentially treatment purposes as well.